Colicin Immunity Protein



Immunity proteins against Colicins (EcCIP) are produced by E. coli alongside the relevant colicin protein (EcCol) to protect the cell from the cytotoxic domain of the colicin. Usually this involves binding to and blocking the active site of the domain, to prevent it from targeting the cells own mechanisms.

Once released from the producing cell, the immunity proteins are no longer needed, as the cytotoxic domain needs to be active once a target cell has been penetrated. Often it is disassociated from the colicin upon binding to an outer membrane receptor on the target cell.

Directed evolution and Colicin7/Immunity-proteins complexes
 Iterative rounds of random mutagenesis and selection of immunity protein 9 (colored yellow) toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to significant increase in affinity and selectivity. Several evolved variants were obtained. The crystal structures of the two final generation variants R12-2 (3gkl; T20A, N24D, T27A, S28T, V34D, V37J, E41G, and K57E) and R12-13 (3gjn; N24D, D25E, T27A, S28T, V34D, V37J, and Y55W) in complex with ColE7 were solved.

Structural alignment of the immunity protein 9 (Im9, 1bxi, colored yellow), evolved variant R12-2 (lime), and immunity protein 7 (Im7, 7cei, colored blue) reveals their structural identity. However, when the immunity proteins-bound colicins within their complexes were aligned, they demonstrate somewhat different picture. The Im9 and Im7 are differ more in their binding configurations (19°, with Tyr54-Tyr55 as the pivot), while the variant R12-2 is in an intermediate configuration between Im9 and Im7. Of note, in the variant R12-2 (3gkl) and Im9 (1bxi) there are Tyr54 and Tyr55, while in the Im7 (7cei) Tyr55 and Tyr56 are homologous to them. The most prominent differences are in the loop between helices α1 and α2 in Im9 (yellow, labeled in black) and evolved variant R12-2 (lime, labeled in black). This loop consists of three mutations: N24D, T27A, and S28T in variant R12-2. We can see the deviations in the relative position of helices α1 and α2, in the loop's backbone and in the side chains of residues 24, 26 and 28.

Comparison of the different Im-colicin complexes reveals changes in the binding configuration of the evolved variants which increase affinity toward ColE7 by re-aligning pre-existing Im9 residues. Glu30 of Im9 (1bxi, colored yellow) forms double salt bridge with Arg54 of ColE9 (orange), whereas Asp51 have not direct side chain–side chain interactions. Asp31 of Im7 (blue) (corresponding to Im9 Glu30) is involved in cluster of salt bridge bonds to Arg520 and Lys525 of ColE7 (darkmagenta), while Asp52 of Im7 (corresponding to Im9 Asp51) is within hydrogen bond distance to <font color='darkmagenta'>Thr531 and Arg530 of ColE7. <font color='lime'>Glu30 in the variant R12-2 (lime) is shifted and forms a <scene name='3gkl/Active_site/8'>double salt bridge to <font color='magenta'>Arg520 of ColE7 (magenta). <font color='lime'>Asp51 is within hydrogen bond distance to <font color='magenta'>Thr531 of ColE7. However, the side chains of <font color='magenta'>Lys525 and Arg530, which are very important in salt bridge contacts with Glu30 and Asp51, respectively, in the structure of the ColE7–Im7 complex have a different conformation that eliminates these contacts in evolved variant R12-2.

In the <scene name='3gkl/Mut/2'>Im9 <font color='magenta'>Val37 (colored magenta) forms stabilizing hydrogen bond with Leu33. In the <scene name='3gkl/Mut/3'>evolved variant R12-2, <font color='darkmagenta'>Ile37 (colored darkmagenta) interacts with two additional residues, Tyr54 and Ser50. Moreover, <font color='darkmagenta'>Ile37 also forms additional hydrogen bond with Gly41 and can thereby have enabled the appearance of the selectivity mutation E41G.

In contrast to the <font color='lime'>evolved variant R12-2 (3gkl), the <font color='cyan'>evolved variant R12-13 (3gjn) carries the <scene name='3gkl/Align/10'>Tyr55Trp mutation in the conserved region. Both <font color='lime'>Tyr55 in R12-2 and <font color='cyan'>Trp55 in R12-13 could sustain the hydrophobic core and create a <scene name='3gkl/Align/11'>hydrogen bond to Lys528 backbone (3gkl colicin residues are colored in <font color='magenta'>magenta, 3gjn colicin residues are colored <font color='blueviolet'>blueviolet ). However, the additional bulkiness of the Trp contributes in expanding its <scene name='3gkl/Align/9'>hydrophobic interactions to Phe541 and Phe513 also leading to the small shift in the alkyl chain of Arg530.

The <scene name='3gkl/Ali/1'>overall conformation of the two evolved variants <font color='lime'>R12-2 (3gkl) and <font color='cyan'>R12-13 (3gjn) is very similar. The variant <font color='lime'>R12-2 carries <scene name='3gkl/Ali/2'>mutation E41G. In the bound wildtype Im9 (yellow) Glu41 makes a <scene name='3gkl/Ali/3'>salt bridge with the <font color='orange'>ColE9’s Lys97 (1bxi). While in the <font color='blueviolet'>R12-13 /<font color='cyan'>ColE7 complex the <font color='blueviolet'>closest ColE7 residues <scene name='3gkl/Ali/4'>contacting <font color='cyan'>R12-13 Glu41 are <font color='blueviolet'>Thr531 (3.37Å) and Lys528 (8.85Å) (3gjn). In the <font color='lime'>R12-2 /<font color='magenta'>ColE7 complex the <scene name='3gkl/Ali/5'>closest <font color='magenta'>ColE7 residue to <font color='lime'>R12-2 Gly41 is <font color='magenta'>Thr531 (9.48Å) (3gkl).

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Colicin-D immunity protein
1tfk, 1tfo, 1v74 - EcCIPD + EcColD catalytic domain

Colicin-E2 immunity protein
2no8 – EcCIPE2 – NMR<BR /> 2wpt – EcCIPE2 + EcColE9 (mutant)

Colicin-E3 immunity protein
3eip – EcCIPE3<BR /> 1jch – EcCIPE3 + EcColE3<BR /> 2b5u – EcCIPE3 + EcColE3 (mutant) <BR /> 1e44 – EcCIPE3 + EcColE3 nuclease domain

Colicin-E5 immunity protein
2dfx – EcCIPE5 + EcColE5 C-terminal domain

2fhz – EcCIPE5 + EcColE5 residues 74-180

Colicin-E7 immunity protein
1ayi, 1unk, 1cei – EcCIPE7<BR /> 2k0d - EcCIPE7 - NMR

1mz8, 7cei – EcCIPE7 + EcColE7 nuclease domain 2jaz, 2jb0, 2jbg, 1znv – EcCIPE7 + EcColE7 nuclease domain (mutant) <BR /> 2erh, 1ujz – EcCIPE7 (mutant) + EcColE7 (mutant)

Colicin-E8 immunity protein
1gxg, 1gxh – EcCIPE8 - NMR<BR />

Colicin-E9 immunity protein
1e0h, 1imp, 1imq – EcCIPE9 - NMR<BR /> 2k5x, 1emv, 1bxi – EcCIPE9 + EcColE9 DNase domain

2vln, 2vlo, 2vlp, 2vlq - EcCIPE9 + EcColE9 DNase domain (mutant) <BR /> 2gze, 2gzg, 2gzi, 2gzj, 2gzf, 2gyk, 1fr2 - EcCIPE9 (mutant) + EcColE9 DNase domain

3gjn – EcCIPE9 (mutant) + EcColE7 nuclease domain

3gkl – EcCIPE9 (mutant) + EcColE7 nuclease domain (mutant)

Colicin-M immunity protein
2xgl – EcCIPM periplasmic domain